This invention relates to a method and an apparatus for measuring membrane characteristics of vesicles, such as the permeability coefficient to a lipophilic ion, the membrane potential, etc. by the employment of an electrode selectivity responsive to the lipophilic ion.
The vesicles mentioned in this invention are structural members which have a membrane permitting the permeation therethrough of a lipophilic ion enclosed with the membrane and a liquid capable of disolving the lipophilic ion, and including living cells, such as an animal cell, a plant cell, a bacterial cell, etc., organellas, such as mitochondria, chloroplast, etc. and artificially made vesicles such as liposome.
Recently many studies are being very actively done for the prescience of various diseases, identification of pathogenes, clarification of the effect of drugs on the biomembrane or screening of new bioactive substances by utilizing various functions of a biomembrane, especially, the transport capacity of the biomembrane.
Methods that have heretofore been employed for measuring the transport capacity of the biomembrane are such as follows: If a vesicle is put in a solution of a substance originally present in a living body, for example, glucose, the glucose flows in and out of the vesicle and, at the same time, water also flows in and out of the vesicle to increase and decrease its volume. By measuring the change in the light scattering intensity or turbidity of the cell suspension which accompanies the volume change of the vesicle, its permeability to the substance (glucose) is detected. In the case of erythrocytes, the permeability of the erythrocytic membrane of the substance is measured by detecting the time when water enters the erythrocytes to burst them to make the solution red, that is, how long it takes to cause hemolysis. Another method is to label the substance by isotope and measure the isotope having passed through the cell membrane, whereby to obtain the permeability of the cell membrane to the substance.
The radioisotope method is the most reliable but incapable of successive measurement and the radioactive material is harmful and should be handled very carefully; therefore, this method is not suitable for measurement of many samples. Since the method of measuring the volume change in the cell which results from the inflow and the outflow of water requires an inflow and an outflow of the substance in a large amount sufficient to deform the membrane, data obtained by this method do not often agree with the values measured by the radioisotope method. The substances used for conventional measurements of the permeability thereto of vesicles are almost those originally contained in the living cells to be measured or the circumstances in which the cells exist. Further, the conventional measurement of the permeability of the vesicular membrane is a mere measurement and the measured values are not utilized for prescience of diseases and like purposes.
An apparatus for measuring the membrane potential of a vesicle is set forth, for example, in "Membrane Potential Measurement Using Lipophilic Ions for Vesicles Directly Unmeasurable with Small Electrodes" by Makoto Muratsugu et al, Digests of Lectures, Second Symposium on Interaction between Biomembranes and Drugs, 1978. This apparatus employs an electrode selectively responsive to a lipophilic ion but is an apparatus for basic researches which is intended only to obtain membrane potentials of vesicles which have large membrane potentials, such as mitochondria, bacteria, etc. A method for measuring the membrane potential with this apparatus is to detect a potential difference .DELTA.E between steady potentials of an electrode in a solution of lipophilic ions before and after the injection of vesicles into the solution and calculate the membrane potential of the vesicle by substituting into a complicated equation the potential difference value .DELTA.E, the volume value of the vesicle and the volume value of the solution suspending the vesicle. With this apparatus, since the steady potential of the electrode is measured after the injection of the vesicle into the solution, as mentioned above, the measurement must be retarded until the electrode potential becomes steady after the injection of vesicles. Generally, it takes time for the electrode potential to become steady after the injection of vesicles into the solution. Especially blood cells of human beings are small in both the membrane potential and the permeability coefficient; in particular, at low temperatures between 0.degree. to 25.degree. C., the permeating speed is low, so that it takes as long as more than 30 minutes for the electrode potential to become steady after the blood cells are injected into the solution. Such a conventional method of measurement requires a lot of time to measure the electrode potential difference .DELTA.E and involves troublesome proceedings of calculating the membrane potential of the vesicle by substituting into a complicated equation the electrode potential difference .DELTA.E measured for each sample, as described above. Therefore, it is impossible to measure the membrane potentials of a large number of samples in a clinical examination.
The permeability coefficient of the vesicular membrane to the lipophilic ion bears a close relation to the membrane fluidity of vesicles; the membrane potential is a difference between potentials on both sides of the vesicular membrane and a physical quantity which serves as a driving force for the ion transfer. The membrane potential and the membrane fluidity of vesicles are both dependent only upon the characteristics of the vesicular membrane and considered as basic factors which define the transport capacity of the vesicular membrane. If valuable information on the transfer of a substance through the vesicular membrane is obtained, it is possible to achieve, for example, the prescience of various diseases, identification of pathogenes, clarification of the effect of drugs on the biomembrane or screening of new bioactive substances.